Schematic representation of the PCR DNA fragments ThyA-F, ThyA-Δ3′, ΔThyA and X-thyAΔ3′-X′

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in "</p><p>Nucleic Acids Research 2005;33(6):e59-e59.</p><p>Published online 30 Mar 2005</p><p>PMCID:PMC1072810.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () PCR amplification using primers thyA-F1 and thyA-R1 () amplifies a 1470 bp fragment (thyA-FL) of the gene containing the promoter, Factor Sigma 54 binding site and the transcription termination flanking the start and stop codons for translation. The 1124 bp product, thyA-Δ3′, amplified using primers thyA-F1 and thyA-R3 (), lacks 347 bp 3′ of the transcription terminator. ΔthyA is a 744 bp product generated by overlapping PCR (see Materials and Methods for details) with most of the coding sequence deleted (+123 to +848; relative to the translational start site), and Δ in the diagram represents the deleted region. X-thyAΔ3′-X′ is a fusion product amplified using primers ColX-thyAF and ColX-thyAR with 5′ and 3′ overhang sequences for the regions of the gene for homologous recombination. () Schematic representation of the strategy for the generation of the ColX-G18D fragment. Two PCR products were first generated using primer sets, ColX-F/ColX-mR and ColX-mF/ColX-R. These products, containing complementary sequences from primers ColX-mF and ColX-mR, were used in an overlapping PCR to produce ColX-G18G using primers ColX-F and ColX-R. () Schematic representation for the amplification of a PCR product from a plasmid, pG18D-Flag, using primers ColX-F and ColX-R. pG18D-Flag contains mutations (bold and underlined) around the signal peptide cleavage site of collagen X, and a Flag sequence inserted at the 3′ region of exon 2 (shaded region)